Hosuk:LabNotes/2013-7-9: Difference between revisions

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*2hr, 4hr were not much different!
*2hr, 4hr were not much different!
*After striping Cy3 probe, there was no signal from Cy3 channel.
*And after re-hybridizing Cy5 probe, rolonies were nicely seen through Cy5 channel.
*Rolonies were pretty stable.





Latest revision as of 00:23, 10 July 2013

Rolony, 13th[edit]

  • Check point
    1. 2hr vs. 4hr CircLigase II reaction
    2. Strip and re-hybridize Cy5 probe


  • Start at 06/29
    • Use cell dishes fixed at 07/07 --> Cells were not plenty… need to thaw new one
    • GC, lysine coated dish
    • Run RT : 5:00pm 07/07 ~ 9:00am 07/08 --> ~15hr.
    • Run CircLigase II : S1 --> 1:30pm 07/08 ~ 3:30pm 07/08 --> 2hr.
    • Run CircLigase II : S2 --> 11:30am 07/08 ~ 3:30pm 07/08 --> 4hr.
    • Run RCA : 4:30 pm 07/08 ~ 12:30pm 07/09 --> ~20hr.


Result[edit]

  • Not as many as before
    • It could be something wrong while fixing (10% formalin once) or cells were not good.
    • For the next time, I’ll do twice fixation. (37% and then 10% formalin), and cells need to be full
    • Rolonies were found inside nucleus more than other area. --> it might cause from not many cells or one time fixation.


  • 2hr, 4hr were not much different!
  • After striping Cy3 probe, there was no signal from Cy3 channel.
  • And after re-hybridizing Cy5 probe, rolonies were nicely seen through Cy5 channel.
  • Rolonies were pretty stable.


  • Strip protocol:
    1. 80% formamide in 2x SSC heated in 60C oven for 5min
    2. Add 200uL of 80% formamide on cells, incubate at RT for 10min.
    3. Wash with H2O twice, and store in PBS


2hr vs. 4hr CircLigase II[edit]

File:S1-2hr S2-4hrCirc 20x-NA0.8 3uLPhi29 exp0.1 EM40.png


After strip[edit]

File:S2-4hrCirc AfterStrip 20x-NA0.8 3uLPhi29 exp0.1 EM40.png


After re-hybridizing Cy5 probe[edit]

File:S2-4hrCirc AfterStripAndReHybriCy5 20x-NA0.8 3uLPhi29 exp0.1 EM40.png